Total Collaborative Study Protocol_Solus One Salmonella v1 1

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1.29  Prepare the Washing Buffer by adding the contents of a single Washing Buffer Activator  sachet and Washing Buffer bottle (60 mL) to 1440 mL or a single Washing Buffer  Activator sachet and Washing Buffer bottle (10 mL) to 240 mL of deionized water to  prepare the washing buffer at assay concentration. Mix until the activator has fully 

dissolved. 

1.30  Leave the first well in the strip empty to serve as a “blank” for measuring the 

absorbance of the substrate. 

1.31  Aliquot 0.1 mL of the Negative Control in the second well and 0.1 mL of the Positive  Control in the third well. Add 0.1 mL of each heated sample to each consecutive well in  the strip. If there are wells left over at the end of a test strip the Positive or Negative 

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Controls may be repeated. 

1.32  Incubate the plate (containing the strips) at 37 ± 1°C for 30 ± 5 min. 

1.33  Following incubation, aspirate the contents of the wells, removing as much of the liquid  as possible. Wash the wells 5–7 times with Washing Buffer ensuring completed filling  and emptying of the wells through each wash cycle. Note: The washing technique is  critical to assay performance; hence it is recommended to use a microplate washer.  1.34  After completion of the washing steps, add 0.1 mL of Conjugate to each well, except for  the blank. Upon completion of the addition of the Conjugate, incubate the plate 

(containing the strips) at 37 ± 1°C for 30 ± 5 min. 

1.35  Repeat the aspiration and wash cycles. 

1.36  After completion of the second aspiration and washing steps, add 0.1 mL of TMB  Substrate to each well, including the blank. Upon completion of the addition of the TMB  Substrate, incubate the plate (containing the strips) for 30 ± 5 min at room temperature 

(15–25°C). 

1.37  After incubation, stop the reaction by adding 0.1 mL of Stop Solution to all wells,  including the “blank” well. Note: The Stop Solution will cause any blue color in wells to 

change to yellow. 

1.38  Where applicable read the optical densities within 10 min in a microplate reader using a  450 nm filter (do not use reference filter), or on the Dynex DS2 instrument using the  “Plate Read Only” setting. Inspect the wells before reading for air bubbles and if present  burst with a sterile needle. Zero the reader against the “blank” well before the other 

wells are read. 

1.39  Interpret results as described in 1.27. 

1.40  Test Result Confirmation 

1.40.1 Using the primary enrichment (step 1.1 above), follow steps starting at step 2.2  below (FDA BAM Ch. 5 secondary enrichments) to confirm each test portion, 

regardless of presumptive result.  

1. 40.2 In addition, all enriched test portions will be confirmed using an alternative  confirmation procedure. Each portion will be streaked directly from the primary 

enrichment onto selective agars, as described by BAM Ch. 5.  

1. 40.3 Transfer typical colonies from each confirmation procedure (traditional and 

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alternative) to TSI/LIA slants. Incubate 35  1  C for 24  2h. 

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